Protein phosphorylation is an essential posttranslational modification regulating nearly every aspect of cell biology. Aberrant phosphorylation drives oncogenic processes like uncontrolled cell proliferation, cell migration, and more. Phosphorylation was recently shown to confer an additional layer of regulatory control over ubiquitylation, another critical regulatory posttranslational modification. The goal of this proposal is to develop chemical tools for studying and mapping patterns of aberrant protein phosphorylation in cancerous tissue; specifically, we propose a method to discover the kinases responsible for phosphorylating ubiquitin in cancerous tissue. We observe that phosphorylation sites are located in key structural motifs necessary for ubiquitin to interact with its effectors and that phosphorylation at some sites has been observed exclusively in tumor samples. As such, we hypothesize that ubiquitin phosphorylation modulates ubiquitin structure, modifying its interaction with effector proteins, branching patterns and distribution, and that some phosphorylation sites may be pathologic. Because current methods cannot answer which or how many kinases are capable of phosphorylating a phosphorylation site, Aim 1 proposes to optimize a chemical crosslinker for the specific covalent capture of kinases which interact with a given phosphorylation site. This system presents several theoretical advantages; namely, it could observe degenerate phosphorylation by multiple kinases and crosslinking would be assessed under native concentrations of kinase and with native effectors present. Preliminary data show that we have developed a covalent crosslinker capable of crosslinking kinases to their cysteine mutant protein substrates and a bioorthogonal crosslinker capable of ligating kinases to modified substrate peptide. In Aim 2, we propose to apply this crosslinker to the study of ubiquitin phosphorylation at four sites, identified under physiologic and pathologic conditions. Completion of this proposal will provide a new chemical tool to address a previously unexplored aspect of kinase biology and will shed light on the posttranslational modification of ubiquitin, an essential regulatory protein that is dysregulated in cancer.